Methods of Isolating and Using Descemet's Membrane and Compositions Including Isolated Descemet's Membrane

This application claims the benefit of U.S. Provisional Application Ser. No. 62/747,715, filed Oct. 19, 2018, which is incorporated by reference herein.

Limbal stem cell deficiency is a major cause of corneal blindness in the United States due to the limited treatment options available and poor long-term prognosis. Limbal stem cells are a population of pluripotent cells on the ocular surface that sustain and regenerate the vital, transparent epithelium of the cornea throughout life. In addition to being transparent, corneal epithelium is crucial for maintaining the avascularity of the cornea, protecting the cornea from infection, and maintaining a healthy tear film over the cornea. Loss of limbal stem cells due to chemical or thermal burns; iatrogenic trauma including, for example, overuse of contact lenses, chronic use of glaucoma drops, or ocular surgery; or an autoimmune disease including, for example, Steven Johnson Syndrome or ocular cicatricial pemphigoid, results in an inability to regenerate normal corneal epithelium on the ocular surface. This inability results in devastating pain and blindness due to corneal erosions, scarring, melting, and conjunctivalization.

This disclosure describes methods of preparing a decellularized Descemet's membrane and an isolated Descemet's membrane, methods of using an isolated Descemet's membrane, and tissues prepared using an isolated Descemet's membrane. This disclosure further describes a composition that includes an isolated Descemet's membrane. In some embodiments, the tissues and methods described herein may be used to treat a limbal stem cell deficiency or as an ocular surface bandage.

In one aspect, this disclosure provides a method that includes removing endothelium from a Descemet's membrane of a cornea to provide a decellularized Descemet's membrane and separating the decellularized Descemet's membrane from the stroma of the cornea to obtain an isolated Descemet's membrane.

In another aspect, this disclosure provides a composition that includes an isolated Descemet's membrane, wherein the Descemet's membrane has been decellularized and separated from the corneal stroma.

The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

This disclosure describes methods of preparing a decellularized Descemet's membrane and an isolated Descemet's membrane, methods of using an isolated Descemet's membrane, and tissues prepared using an isolated Descemet's membrane. This disclosure further describes a composition that includes an isolated Descemet's membrane. In some embodiments, the tissues and methods described herein may be used to treat a limbal stem cell deficiency or as an ocular surface bandage.

Several surgical options have been reported for the treatment of severe limbal stem cell deficiency: keratolimbal allografts (KLAL), simple limbal epithelial transplants (SLET), and cultured limbal transplants (CLET). The use of each of these techniques, as further described below, remains limited and suboptimal.

In KLAL the entire limbus and adjacent cornea and conjunctiva (corneolimbal ring) are excised from a donor cornea and the entire chunk of mixed tissues is transplanted on the recipient eye. This procedure results in a high rate of rejection because so many mixed tissues and antigens (including resident antigen-presenting dendritic cells in the limbus) are transplanted in addition to the limbal stem cells. Systemic immunosuppressive drugs must be administered to the patient to prevent rejection. Additionally, two donor corneas are usually required to have enough donor corneolimbal tissue to cover the damaged limbus of a single recipient eye. The procedure is also time-consuming and difficult for the surgeon because the dissection needs to be done in the operating room at the time of surgery (so that the graft is fresh). It can further be challenging to dissect out the donor limbus without injuring the donor stem cells or taking too much of the adjacent cornea and conjunctiva.

In SLET, a KLAL graft (corneolimbal ring) is harvested from a donor eye, but instead of being transplanted directly, the graft is cut into multiple small fragments (limbal explants). These explants are then scattered over the diseased cornea on a bed of amniotic membrane. Over time, limbal stem cells grow out from the fragments and resurface the cornea. The fragments are then removed after the cornea is covered with cells. Compared to KLAL, SLET does not require as much donor tissue and less extraneous tissue, aside from the limbal stem cells, is ultimately transplanted because the limbal fragments are removed. Nevertheless, results are still variable and suboptimal. The limbal fragments often dislodge before there is limbal stem cell outgrowth, resulting in a failed surgery. Also, significant trauma to the donor tissue may occur in preparing the explants, resulting in a significant portion of the fragments demonstrating no outgrowth of limbal stem cells. Prior to transplantation, there is also no way to objectively test the viability of the transplanted stem cells. Finally, the long-term survival of limbal stem cells on the cornea, outside of a limbal niche microenvironment, is limited. Without a limbal niche microenvironment, stem cells have difficulty maintaining their stem cell phenotype and often lose the ability to proliferate indefinitely.

One method of performing CLET is described in U.S. Pat. No. 7,347,875. Generally, in this method, limbal stem cells are removed from the intended recipient or a donor by taking small biopsies (explants) from the limbus of a healthy eye. The explants are then grown in culture on a human amniotic membrane. After a sheet of stem cells has been grown, the entire sheet, including the amniotic membrane, is transplanted onto a recipient eye. With this technique, a pure population of stem cells is transplanted, so the risk of immune rejection is presumed to be lower than with KLAL. Moreover, with CLET, a large number of stem cells may be transplanted from a small piece of donor limbus. Also, the viability of the stem cells in culture can be confirmed before transplantation surgery. Finally, with CLET, the bulk of the graft preparation work is done in the lab, outside of the operating room, saving surgeons time and money.

Several problems nevertheless exist with this method for preparing limbal stem cell grafts. The first is that using biopsies of limbal tissue results in significant trauma to the donor limbus and donor limbal stem cells. As a result, up to 20% of limbal explant cultures fail to grow in the lab. Second, using amniotic membrane introduces another potential exogenous source of infection to the transplant recipient because amniotic membrane is harvested from different donors than the donor used for the limbal biopsies. Therefore, additional risk of viral or bacterial transmission is introduced when using amniotic membrane. Third, though processed amniotic membrane has been shown to be non-immunogenic in most patients, additional antigens from the allogenic placental tissue may still be a risk factor in immune rejection of the transplanted graft. Fourth, the use of amniotic membrane as a substrate for the cell cultures requires a lot of resources; most surgeons do not have access to amniotic membrane-based CLET grafts. Moreover, amniotic membrane is expensive and difficult to store (requiring storage at −80° C. with a limited shelf life). Fifth, amniotic membrane is neither transparent nor perfectly stable on the ocular surface.

Because amniotic membrane is neither transparent nor completely stable on the ocular surface, there are two potential outcomes that occur after an amniotic membrane-based limbal stem cell graft is transplanted on the eye, and neither is ideal. One potential outcome is that the amniotic membrane persists and becomes incorporated on the cornea long-term. The clarity of the patient's cornea is compromised by the semi-opaque amniotic membrane, and vision can remain poor despite successful transplantation of viable limbal stem cells. The alternative outcome is that the amniotic membrane dissolves before becoming fully incorporated into the cornea. In this case, the cornea can be clear, but the transplanted stem cells lose the substrate that helps to sustain their viability and, potentially, their stem cell phenotype. Without a limbal niche microenvironment, it is very difficult for stem cells to maintain their stem cell phenotype. Stem cells growing directly on the cornea itself, outside a normal limbal niche microenvironment or limbal niche-like environment, will eventually differentiate into mature corneal epithelial cells and lose their ability to proliferate indefinitely. This differentiation results in long-term failure of the limbal stem cell transplants as terminally differentiated epithelial cells cannot sustain the ocular surface throughout the patient's lifetime. Consequently, long-term success (for example, beyond five years) remains limited with current amniotic membrane-based limbal stem cell grafts.

As further described herein, in some aspects, this disclosure describes a modified CLET that uses Descemet's membrane as a substrate for culturing limbal stem cells instead of amniotic membrane. Descemet's membrane is the multilayered basement membrane of the corneal endothelium (see). Descemet's membrane is routinely isolated, along with the endothelial cells, from donor corneas and transplanted intraocularly in patients to replaced damaged Descemet's membrane and/or corneal endothelial cells as reported by Park, et al.,2015; 122:2432-2442 (see also U.S. Pat. No. 8,889,415). Transplantation of Descemet's membrane onto the external ocular surface of the eye is not known to have been previously described.

As further described herein, Descemet's membrane unexpectedly provides multiple advantages as a substrate for supporting proliferation and long-term survival of limbal stem cells, both in vivo on the ocular surface and ex vivo in culture. The similarity of Descemet's membrane to the basement membrane of the limbal niche microenvironment, biochemically, is an obscure characteristic of Descemet's membrane that has not previously been exploited to promote the growth of limbal stem cells and to inhibit further differentiation into non-amplifying corneal epithelial cells. The methods of this disclosure exploit this characteristic of Descemet's membrane, and this disclosure describes, in some embodiments, a method of using Descemet's membrane as a substrate for culturing limbal stem cells.

In one aspect, this disclosure describes a method of removing endothelium from a Descemet's membrane of a cornea to provide a decellularized Descemet's membrane. In a further aspect, this disclosure describes separating a decellularized Descemet's membrane from the stroma of the cornea to obtain an isolated Descemet's membrane.

In some embodiments, the cornea may be a donor cornea including, for example, a cadaveric cornea. In some embodiments, the cornea may be a human cornea. In some embodiments, the cornea may be a porcine cornea.

In some embodiments, corneal endothelium may be separated from Descemet's membrane to form a decellularized Descemet's membrane using mechanical, enzymatic, and/or chemical decellularization. In some embodiments, the separation of the corneal endothelium from Descemet's membrane to form a decellularized Descemet's membrane leaves the corneal epithelium intact.

In some embodiments, a decellularized Descemet's membrane may be separated from the stroma of a cornea to obtain an isolated Descemet's membrane by manually peeling the Descemet's membrane from the stroma of the cornea. In some embodiments, the corneal epithelium may preferably be removed with the stroma of the cornea.

In some embodiments, a decellularized Descemet's membrane may be separated from the stroma of a cornea to obtain an isolated Descemet's membrane by injecting air or fluid or both into the cornea. The fluid may include any suitable fluid. In some embodiments, the fluid may include, for example, saline, corneal storage solution, or any buffered solution. In some embodiments, the residual cornea (e.g., including the corneal epithelium and corneal stroma) that has been separated from Descemet's membrane may be excised to expose the isolated Descemet's membrane.

In some embodiments, separating the decellularized Descemet's membrane from the stroma of the cornea and removing that stroma includes exposing the isolated Descemet's membrane to a limbal stem cell found in the corneolimbal ring of the cornea. In some embodiments, the method may further include making a partial-thickness incision in the corneolimbal ring. Such an incision may promote the outgrowth of limbal stem cells from the corneolimbal ring. Drawings of one exemplary method of separating the decellularized Descemet's membrane from the stroma of the cornea and exposing the isolated Descemet's membrane to a limbal stem cell is shown in-.

In other embodiments, the isolated Descemet's membrane may be excised completely from the rest of the cornea (including the corneolimbal ring) including, for example, by trephinating the Descemet's membrane.

In another aspect, this disclosure describes a method of storing an isolated Descemet's membrane. In some embodiments, including, for example, when the Descemet's membrane has been completely excised by trephination or manually peeled from the rest of the cornea, the isolated Descemet's membrane may be preserved without regard to cell viability. In some embodiments, including, for example, when Descemet's membrane has not been completely excised from the corneolimbal ring or when the isolated Descemet's membrane has been used as a cell culture substrate, the isolated Descemet's membrane may be preserved by a means that maintains cell viability. In some embodiments, the isolated Descemet's membrane may be frozen, lyophilized, and/or cryopreserved.

In some embodiments, the isolated Descemet's membrane may be sterilized before or after preservation or both before and after preservation. The isolated Descemet's membrane may be sterilized by any suitable means including, for example, by gamma irradiation, chemical disinfectant, antibiotic treatment, ethylene oxide gas treatment, or supercritical COexposure.

In a further aspect, this disclosure describes methods of using an isolated Descemet's membrane.

In some aspects, this disclosure describes using an isolated Descemet's membrane as a cell culture substrate. For example, in some embodiments, this disclosure describes using an isolated Descemet's membrane as a cell culture substrate to support proliferation of a limbal stem cell. In some embodiments, the limbal stem cell may adhere to the isolated Descemet's membrane.

In some embodiments, an isolated Descemet's membrane may be attached to a cell culture surface including, for example, tissue culture plastic. A drawing of one exemplary method of attaching Descemet's membrane to a cell culture surface is shown in. In some embodiments, the Descemet's membrane may be reversibly attached to the cell culture surface.

In some embodiments when an isolated Descemet's membrane is attached to a cell culture surface, the isolated Descemet's membrane will preferably have been completely excised or manually peeled from the rest of the cornea (including the corneolimbal ring). In some embodiments, including, for example, when an isolated Descemet's membrane is reversibly attached to a cell culture surface, a limbal explant may be cultured in the presence of the isolated Descemet's membrane. In some embodiments, the limbal explant may be from the same donor (for example, from the same cadaveric cornea) or different donor (for example, from a living donor's cornea) as the isolated Descemet's membrane. In some embodiments, the limbal explant includes a part of the corneolimbal ring. In some embodiments, the limbal explant includes an entire corneolimbal ring. In some embodiments, the limbal explant may be cultured under conditions that allow outgrowth of a limbal stem cell from the limbal explant onto the isolated Descemet's membrane, resulting in an isolated Descemet's membrane with limbal stem cells on its surface.

In some embodiments, separating the decellularized Descemet's membrane from the stroma of the cornea to obtain an isolated Descemet's membrane exposes the isolated Descemet's membrane to limbal stem cells found in the corneolimbal ring. For example, when the isolated Descemet's membrane is not excised (by trephination) or manually peeled from the corneolimbal ring, the isolated Descemet's membrane may be exposed to limbal stem cells found in the corneolimbal ring by removing the cornea stroma. In some embodiments, the corneolimbal ring remains attached to the isolated Descemet's membrane.

Additionally or alternatively, an isolated Descemet's membrane may be exposed to limbal stem cells obtained from another source or obtained from the same donor source but separated from the corneolimbal ring. In some embodiments, a limbal explant culture may be treated to release limbal stem cells (into a cell suspension) which may be seeded onto an isolated Descemet's membrane. In some embodiments, a limbal explant culture and/or a limbal tissue ring may be digested with one or more of trypsin (Sharifi, et al.,2010; 34:53-55), dispase (Zhang, et al., Curr Eye Res. 2016; 41:318-325), or collagenase (Chen, et al.,2011; 17:537-548) to release the basal limbal stem cells (see). In some embodiments, once in suspension, the limbal stem cells may be seeded onto an isolated Descemet's membrane without any further manipulation (see). In some embodiments, the limbal stem cells may be sorted (for example, using flow cytometry) to select cells with a specific limbal stem cell marker or markers prior to being seeded onto an isolated Descemet's membrane. Exemplary methods of preparing limbal stem cells for culture on Descemet's membrane are described in Example 8A and 8B.

Limbal explant cultures and/or limbal stem cells may be maintained in any suitable growth media. Suitable growth media may include, for example, a growth medium containing human autologous serum, fetal bovine serum, human platelet lysates, and a growth medium containing serum-free medium with bovine pituitary extracts, growth supplement with recombinant components, or chemically defined supplements.

An isolated Descemet's membrane may be cultured under conditions that allow for growth of limbal stem cells (e.g., from a limbal explant and/or from a corneolimbal ring) onto the isolated Descemet's membrane. A drawing of one exemplary method of culturing Descemet's membrane to achieve limbal cell outgrowth onto the Descemet's membrane is shown in. In some embodiments, the method may include making a partial-thickness incision in the corneolimbal ring to promote the outgrowth of limbal stem cells.

In some embodiments, the method may include placing the isolated Descemet's membrane in a cell culture media. The cell culture media may include any suitable cell culture media including, for example, an epithelial cell growth media or other media suitable for corneal organ culture. An exemplary media suitable for corneal organ culture is CorneaMax® (Eurobio, Les Ulis, France). In some embodiments, the cell culture media may be serum free. In some embodiments, including, for example, when the cell culture media is serum free, cell culture media may include pituitary extract. In some embodiments, the cell culture media may include one or more of a complex culture media supplemented with fetal bovine serum, a complex culture media supplemented with human serum, a complex culture media supplemented with platelet lysate serum, or a chemically-defined keratinocyte growth media. In some embodiments, the cell culture media may preferably promote limbal stem cell growth and/or maintain limbal stem cell pluripotency.

In some embodiments, the method may include incubating the isolated Descemet's membrane in the cell culture media under typical cell culture conditions including, for example, at a temperature in a range of 32° C. to 38° C. and/or at 5% CO. In some embodiments, the isolated Descemet's membrane is incubated with a limbal explant and/or a limbal stem cell.

In an additional aspect, this disclosure describes methods of transplanting an isolated Descemet's membrane to an ocular surface of a patient in need thereof.

In some embodiments, the transplanted Descemet's membrane may be decellularized. In some embodiments, the transplanted Descemet's membrane may include limbal stem cells. A drawing of one exemplary method of transplanting a Descemet's membrane including limbal stem cells is shown in. In some embodiments, the transplanted Descemet's membrane may include an isolated Descemet's membrane prepared by any of the methods described herein. For example, an isolated Descemet's membrane including limbal stem cells produced using the methods described herein may be transplanted to a patient exhibiting a partial limbal stem cell deficiency, a total limbal stem cell deficiency, a persistent epithelial defect, an epithelial erosion, a corneal ulcer, a corneal melt, and/or an ocular surface disease. Limbal stem cell deficiency may be caused by, for example, burns (chemical, thermal, or industrial); excessive use of certain medications; iatrogenic trauma, including, for example, overuse of contact lenses, chronic use of glaucoma drops, or ocular surgery; an autoimmune disease including, for example, Steven Johnson Syndrome or ocular cicatricial pemphigoid; or graft-versus-host disease (GVHD); etc.

In some embodiments, transplanting an isolated Descemet's membrane to an ocular surface of a patient, as described herein may have certain advantages over existing treatments for limbal stem cell deficiency. For example, transplanting an isolated Descemet's membrane to an ocular surface of a patient, as described herein (e.g., including a limbal stem cell), has certain advantages over an amniotic membrane-based CLET graft. A CLET-like procedure performed using an isolated Descemet's membrane is less expensive than an amniotic-membrane based method (because it takes advantage of the free Descemet's membrane that comes with every donor cornea and limbus), and it also makes it possible to produce CLET-like grafts in an eye bank setting, improving surgeon access to CLET technology.

One of the major challenges of limbal stem cell transplantation is sustaining the long-term viability, proliferative potential, and overall stem cell phenotype of the transplanted cells outside of the native limbal niche microenvironment. As shown inand Example 2, when used as a substrate, Descemet's membrane promotes rapid expansion of limbal stem cells in culture while maintaining the stem cell phenotype in the majority of cells out to several weeks in culture (based on cellular morphology and the expression of stem cell markers ABCG2 and ΔNp63α. Transplantation of Descemet's membrane along with limbal stem cells on the ocular surface provides a novel, viable technique for establishing a stable niche-like microenvironment on the cornea, and thus may provide a robust and promising technique for long-term reconstruction of the ocular surface. Additionally, intact Descemet's membrane is optically clear (see), resistant to collagenase digestion (see), minimally immunogenic when transplanted intraocularly, and freely available with every donor cornea from which stems cells are harvested.

In some embodiments, including, for example, when the isolated Descemet's membrane is decellularized, the isolated Descemet's membrane may be used as an ocular bandage including, for example, in patients with an ocular surface trauma, a recurrent erosion, a corneal melt, or a sterile corneal ulcer. Such patients may include any patient with a disease that compromises the ocular surface including, for example, patients with diabetes, patients with glaucoma, patients with injuries resulting from chronic contact lens wear, patients with complications of LASIK surgery, patients with neurotrophic concerns, patients with severe tear deficiency, and/or patients with exposure keratopathy.

For example, an isolated Descemet's membrane (with or without the presence of a limbal stem cell) may be useful as a reconstructive therapy in cases of minor ocular surface burns or trauma. For example, when the epithelium is damaged but enough of the native limbal stem cell population remains to regenerate the corneal epithelium, isolated Descemet's membrane (without a limbal stem cell), when placed as a resurfacing treatment, may establish a stable bandage layer on the ocular surface of the cornea that promotes regrowth of the patients' own epithelium in situ, while protecting the corneal stroma from enzymatic degradation by inflammatory mediators in the tear film due to its natural resistance to collagenase. Isolated Descemet's membrane can remain stable over long periods of time but is still transparent and visually unobstructive. Since the isolated Descemet's membrane is decellularized to remove endothelial cells, the isolated Descemet's membrane has no cells for the body to reject making the risk of rejection minimal.

Isolated Descemet's membrane may be adhered to the recipient cornea by any suitable means. In some embodiments, the Descemet's membrane may be adhered to the recipient cornea by air drying. In some embodiments, the Descemet's membrane may be adhered to the recipient cornea using one or more of a fibrin sealant, colloidal silica nanoparticles, and light-initiated rose bengal collagen cross-linking.